Easy access to tumor samples, without the need for painful and expensive tumor biopsies, would allow clinicians and researchers to repeatedly interrogate patient samples to identify mechanisms of therapeutic resistance and personalize subsequent treatment strategies to these continually evolving tumors. The great hope in circulating tumor cell (CTC) research lies in the potential for these rare cells to be accessible va a fluid biopsy that would permit frequent, minimally invasive sampling of tumor cells for the same molecular assays performed on traditional biopsies. Furthermore, access to CTCs would enable many critical future studies, increasing our understanding of the metastatic cascade, tumor heterogeneity, drug resistance, etc. In the proposed research, a new analysis technique termed Vertical Exclusion-based Rare Sample Analysis (VERSA) will be developed. The VERSA combines CTC purification with downstream protein analysis, RNA extraction, and DNA extraction, all on a single chip from a single patient sample. The VERSA will enable multi- endpoint analyses on CTCs, providing users with a comprehensive snapshot of CTC function at a molecular level. The fundamental technology underlying the function of the VERSA is exclusion-based sample preparation, a new method for performing isolations in which magnetic beads are used to selectively bind an analyte of interest and then draw the captured analyte through an oil barrier and into a second aqueous liquid. Importantly, since exclusion-based purification is achieved by simply drawing an analyte from the sample to another aqueous buffer, the original sample is never diluted or washed away. This enables the sequential extraction of multiple analytes from a single sample including protein, RNA, and DNA. Development and testing of the VERSA-based platform with patient samples will occur throughout three Specific Aims. In the first Aim, we will optimize and validate two additional readouts, subcellular protein localization and total protein quantification. In Aim 2, we will combine CTC isolation with subsequent isolation of both mRNA (for RT-PCR readouts) and DNA (for SNP and sequencing readouts). In Aim 3, we will integrate and validate the readouts of Aims 1 and 2 into a single chip and use this platform to perform a multi-endpoint prospective clinical trial on 40 patients with prostate cancer. During this trial, we will collect correlated protein, gene expression, and genomic data describing the status of the androgen receptor (AR), the major therapeutic target in prostate cancer. This trial will lay the groundwork for futur biological and clinical studies with the VERSA platform.